I´m trying to amplify two exons of CYP2D6, but I have been obtaining inespecific bands. I have aligned the sequences of CYP2D6 and CYP2D7 (a pseudogene with similar sequence) with my primers and they perfectly align with both sequences, so I suppose I've been amplifying both sequences, what should I do? I need to sequence just CYP2D6 exons.
I agree with Mr.Laurence however another way is that you can use nested PCR to get the correct fragment. On this concern you will need to design two primer sets and then go to the two step PCR , please check nested PCR technique on line it is simple and easy. In my experment I have faced the same trouble and nested PCR worked very well with me.
Good luck
ar your primers designed in non coding dna or introns.This often distinguishes between gene and pseudogene
Hi, Renzo,
http://www.pnas.org/content/87/6/2206.full.pdf
this paper may help. Wishes!
I would clone the purified fragment and then subject your clones to restriction digestion. I assume they can distinguished this way. Then fully sequence the one with the correct digest pattern
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